TNF-α Suppresses Apelin Receptor Expression in Mouse Quadriceps Femoris-Derived Cells

Abstract

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.

Keywords: TNF-α; aging; apelin receptor (APJ); skeletal muscle.

Figure 1

Muscle mass of the quadriceps of young and old mice. (A) Body weight (g), (B) muscle mass (mg), and (C) muscle mass (mg)/body weight (g) in young (3-month-old) and old (24-month-old) mice. Body weight was significantly higher in old mice than in young mice (p < 0.001; (A)). Muscle mass did not significantly differ between young and old mice (p = 0.481; (B)). However, muscle mass/body weight was significantly lower in old mice than in young mice (p < 0.001; (C)). Data are expressed as the mean ± standard deviation (SD). Asterisks indicate p < 0.05.

Figure 2

Relative expression of Apelin, and Apj mRNA in the quadriceps femoris. (A) Apelin and (B) APJ expression in young (3-month-old) and old (24-month-old) mice. There was no difference in Apelin mRNA expression between young and old mice (Figure 1A). Apj mRNA expression was significantly lower in old mice than in young mice (p < 0.001, Figure 1B). Data are expressed as the mean ± standard deviation (SD). Asterisks indicate p < 0.05.

Figure 3

APJ−positive cells in the quadriceps femoris. (A,B) Dot plot analysis of CD45-negative myogenic cells. X-axis, Sca-1; Y-axis, APJ. (C,D) Ratio of APJ-positive cells among CD45-negative/Sca1-negative cells © and CD45-negative/Sca1-positive cells (D) in young (3-month-old) and old (24-month-old) mice. Significant reduction in APJ-positive cells was found in Sca1+ (p = 0.016) and Sca1− cells (p = 0.016). Data are expressed as the mean ± standard deviation (SD). Asterisks indicate p < 0.05 (Mann–Whitney U test).

Figure 4

Tnfa mRNA and TNF-α protein expression in the quadriceps femoris. (A) Tnfa mRNA levels in young (3-month-old) and old (24-month-old) mice. (B) Image of a Western blot showing TNF-α protein expression relative to GAPDH at three (3M) and 24 months (24M). (C) TNF-α protein levels in the quadriceps femoris of young (3-month-old) and old (24-month-old) mice. Significant elevation of TNF-α mRNA and protein levels was found in the old group. Data are expressed as the mean ± standard deviation (SD). Asterisks indicate p < 0.05.

Figure 5

Effect of TNF-α on Apelin and Apj expression. Relative expression of Apelin (A,B) and Apj (C,D) in young and old mice-derived muscle cells after stimulation with 0 (control), 2.5, or 25 ng/mL mouse recombinant TNF-α for 8 and 24 h. Significant elevation of Apelin mRNA expression was only observed in young mice-derived muscle cells after TNF-α stimulation for 8 and 24 h (A,B). Apj mRNA expression was significantly reduced following TNF-α stimulation for 8 and 24 h in both young and old mice-derived muscle cells (C,D). Data are expressed as the mean ± standard deviation (SD). Asterisks indicate p < 0.05.